Themed Section: Science and Technology
Anti-Inflammatory Activity of Alkaloids from Murraya Koenigii
Leaves In Animal Models
Rupali Patil
Department of Pharmacology, Gokhale Education Society’s Sir Dr. M. S. Gosavi College of Pharmaceutical Education & Research, Nasik, India
ABSTRACT
Alkaloids have a wide range of pharmacological properties, including anti-inflammatory activity. The purpose of the present study was to investigate the anti-inflammatory and antioxidant activity of Murraya koenigii leaves. The hind paw edema was produced in rats by subplantar injection of Carageenan. Pet ether extract (PMK) of Murraya koenigii leaves and alkaloids (AMK) isolated from PMK at doses of 100 and 300 mg/kg/day, p.o. were given for 11days to observe % inhibition of paw edema which was comparable with Aspirin (100 mg/kg, p.o.) used as a reference drug. PMK and AMK produced a significant (p<0.05) inhibition of paw edema. PMK and AMK treatment significantly reversed the Carageenan induced decrease in paw Superoxide dismutase (SOD), Catalase (CAT), reduced glutathione (GSH) levels as compared to Carageenan treated rats. Lipid peroxidation (LPO) induced by Carageenan treatment was significantly reversed after administration of PMK and AMK. hematological analysis of carageenan-treated rats exhibited significant (p<0.05) decrease in RBC count, Hb content and PCV after treatment with aspirin, compared to the control. PMK or AMK treated animals showed normal erythrocyte (RBC) count, hemoglobin (Hb), packed cell volume (PCV), near to control group. The total leukocyte (WBC), lymphocyte, neutrophils, and basophils count were higher in rats treated with aspirin compared to control. PMK and AMK treatment showed decreased platelet count compared to control. The significant reductions observed in the activity of ALT and AST in PMK and AMK treated animals compared to control.
Keywords : Inflammation, Carbazole Alkaloid, Carageenan, MurrayaKoenigii
I.
INTRODUCTIONInflammation is an essential protective process preserving the integrity of organisms against physical, chemical and infective insults which frequently and erroneously leads to the damaging of normal tissues (Serhan and Levy 2003). The process of inflammation is characterized by increased vascular permeability at inflamed site followed by localization and margination of neutrophils. An acute inflammatory process is comprised of inflammation mediators including neutrophil-derived reactive oxygen species (ROS), nitric oxide (NO-) (Syahida et al. 2010; Valko et al.
2006), prostaglandins (PGs), and cytokines (FitzGerald and Patrono 2001). Oxidative mechanisms are
reported at the origin of inflammation and ROS such
as superoxide anion, hydroxyl radical and peroxynitrite participate in the process of inflammation in various tissues and has suggested the
use of antioxidant substances (Trenam 1992; Bermond
1989). Therefore, compounds that have scavenging
NSAIDs, including cyclooxygenase (COX)-2 inhibitors, exhibit anti-inflammatory effect through inhibition of COX and are among the most widely prescribed medications for the clinical treatment of inflammatory diseases such as arthritis, lumbago and rheumatism. However, NSAIDs have been associated with gastrointestinal (GI) toxicity. The cardiovascular (CV) toxicity is associated with COX-2 inhibitors (Scheiman and Hindley 2010). Numerous clinical observations have associated the use of aspirin with blood disorders like anemia and cytopenias (Raybak, 1992). In this context, there arise new scopes for Herbs and Herbal Formulation in treatment of inflammatory disorders.
Murraya koenigii L. (Rutaceae), commonly known as curry tree, is a tropical to sub-tropical tree native to India. Traditionally the plant is used as tonic, stomachic and carminative (Kirtikar and Basu 1993). Methanolic extract of Murraya koenigii leaves is known to possess analgesic and anti-inflammatory activity (Gupta et al. 2010). Murraya koenigii contains carbazole alkaloids (Narasimhan et al.1968; Chowdhury and Chakraborty 1971) having antioxidant (Rao et al. 2006, Arulselvan and Subramanian 2007; Gupta and Sharma 2010) anti-inflammatory, anti-tumour (Muthumani et al.2009), anti-trypanocidal ( Das and Chakraborty 1965) and mosquitocidal activities (Chakrabarty et al.1997).
The present research was aimed to evaluate the anti-inflammatory and antioxidant activity of pet ether extract of Murraya koenigii L. leaves (PMK) and total alkaloids separated from pet ether extract of Murraya koenigii leaves L. (AMK). The anti-inflammatory activity of PMK and AMK was evaluated using carrageenan induced rat paw edema (Winter et al. 1962). The antioxidant activity revealed by in vitro radical scavenging assays is further confirmed by evaluating cellular antioxidant defense system. The study was further extended to assess effect of PMK
and AMK on haematological profile of experimental animals.
II.
METHODS AND MATERIAL1) Animals
Albino Wistar rats (100-150 g) of either sex were used for this study. The animals were housed at 24 ± 2°C and relative humidity 55 ± 5 with 12:12 h light and dark cycle. They had free access to food and water ad libitum. The animals were acclimatizated for a period of seven days before the study. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of MGV’s Pharmacy College, Nasik.
2) Drugs and chemicals
Carrageenan and 1, 1-diphenyl-2-picryl hydrazyl (DPPH) (Sigma-Aldrich, MO, USA), aspirin (Research Lab, Mumbai) were used for this study.
3) Plant material and extraction
Leaves of Murraya koenigii (1 Kg) were purchased from the local market and were identified by Dr. P.G. Diwakar, Jt. Director, Botanical Survey of India, Pune, where a voucher specimen (MUKKID 1) has been retained. The leaves were dried in shade and powdered mechanically. Powdered leaves of Murraya koenigii were defatted with the Petroleum ether (60-80°C). The filtrate was concentrated to get pet ether extract of Murraya koenigii (PMK) (yield: 12.6 % w/w).
The extract was further subjected to isolation of alkaloids according to method of Cordell GA (1981). The aqueous portion was dried to get crude alkaloidal fraction of pet ether extract of Murraya koenigii leaves (AMK) (yield: 48.33 % w/w).
4) Treatment
administration for 11 days exhibit highest antinociceptive activity. Accordingly, PMK (100 and 300 mg/kg) was suspended in 0.1 % CMC and AMK (100 and 300 mg/kg) was dissolved in water and administered orally to animals for 11 days. Control group received 0.1% CMC, p.o., for 11 days.
5) Phytochemical analysis
Phytochemical analysis of PMK was carried out according to methods described earlier (Trease and Evans, 2002).
Identification
UV-visible spectra: AMK revealed peak at 295.0, 256.50 and 246.50 nm when spectra was run using Shimadzu-2450.
FTIR: AMK depicted presence of functional groups like N-H stretch (3394.83 to 3556.85 μ ) Aliphatic C-H stretch superimposed on N-H stretch (2974.33 μ), N-H bend (1610.61), C-N vibration (aromatic, secondary) (1276.92).
6) In vitro Antioxidant activity
In vitro antioxidant activity of PMK was evaluated by DPPH method (Molyneux 2004). Scavenging free radical potential was evaluated against ethanolic solution of DPPH, a stable free radical.
Total antioxidant activity was determined using ammonium thiocyanate method (Mistuda et al. 1996). Scavenging activity against H2O2 (Ruch et al. 1989) was tested to determine ability of PMK to inhibit the formation of hydroxyl radical.
7) Anti-inflammatory activity
On 11th day of experiment, 1 h after test drug administration, pedal inflammation was induced in rats as described by (Winter et al. 1962). A suspension of 0.1 ml of 1% Carrageenan was injected into the sub
plantar tissue of right hind paw of each rat. The paw volume was measured at 0, 1, 2, 3 and 4 h using Plethysmometer (UGO Basile, Italy) (Vogel, 2002). Aspirin (100 mg/kg, p.o.) was used as reference drug.
8) Biochemical estimation in Carrrageenan treated paw tissue:
After measurement of volume, the rat paws were dissected out, immediately washed in ice-cold saline and weighed. A 10 % homogenate was prepared in 0.1 M Tris buffer, pH 7.4. The homogenate was centrifuged at 15,000 g for 20 min. The supernatant was used for estimation of SOD, Catalase, GSH and LPO. Blood was collected by cardiac puncture and used for estimation of hematological parameters like erythrocyte (RBC) count, hemoglobin (Hb), packed cell volume (PCV), total leukocyte (WBC) count, lymphocyte count, neutrophils count, basophils count, glucose, alanine aminotranferease (ALT) and aspartate aminotransferase (AST).
8.1 Superoxide dismutase
The assay of SOD was based on the ability of SOD to inhibit spontaneous oxidation of adrenaline to adrenochrome (Saggu et al. 1989; Misra and Fridivich 1972).The results were expressed as nmol SOD U/mg wet tissue.
8.2 Catalase
The Catalase activity assay was based on the ability of CAT to induce the disappearance of hydrogen peroxide (Beers and Sizer, 1952). The results were expressed as catalase U/g wet tissue.
8.3 Reduced glutathione
8.4 Extent of Lipid peroxidation
LPO as evidenced by the formation of Thiobarbituric acid reactive substances (TBARS) was measured by the method of Niehaus et al., (1968). The results were expressed as LPO nmol/g wet tissue.
9) Statistical analysis
The mean ± SEM values were calculated for each group. One-way ANOVA followed by Dunnett’s multiple comparison tests were used for statistical analysis. Values of P<0.05 was considered statistically significant.
III.
RESULTS AND DISCUSSIONPhytochemical analysis of PMK revealed presence of alkaloids, triterpenoids, flavonoids, tannins and phenols.
In vitro antioxidant study
In DPPH assay, the % scavenging activity increased with the increase in concentration of PMK. The IC50 value was found to be 98.5 (Fig.1). Total antioxidant activity assay and H2O2 assay also showed increased % scavenging activity of PMK with the increase in concentration of the extract. The IC50 value was found to be 101.7 ppm (Fig.2) and 99.5 ppm (Fig.3) respectively.
Anti-inflammatory activity
Carrageenan treated rats showed gradual increase in paw volume from 1-3 h while the paw volume was decreased at 4th h. Treatment with PMK and AMK (100 and 300 mg/kg/day, p.o., for 11days) significantly (p<0.05) inhibited the extent of carageenan induced paw edema at 1st and 3rd h. Maximum inhibition was observed with AMK (300), 71.87%, at 3rd h after carageenan administration. Aspirin exhibited 72.91% inhibition at 3rd h.
Biochemical estimations in Carrrageenan treated paw tissue
Carageenan treated rats showed decreased levels of SOD, CAT and GSH in paw tissue homogenates. Administration of PMK and AMK (100 and 300 mg/kg/day, p.o., for 11days) significantly reversed the Carageenan induced decrease in paw SOD, CAT and GSH levels as compared to Carrageenan treated rats. On the other hand, Carrageenan treatment induced lipid peroxidation, as indicated by significant high MDA levels in inflamed paw tissue. Administration of PMK and AMK (100 and 300 mg/kg/day, p.o., for 11 days) significantly reversed the extent of lipid peroxidation as compared to Carrageenan treated rats. Further, hematological analysis of carrageenan-treated rats exhibited significant (p<0.05) decrease in RBC count, Hb content and PCV after treatment with aspirin, compared to the control. PMK or AMK treated animals showed normal RBC count, Hb content and PCV near to control group. The WBC count, lymphocyte count, neutrophils count, basophils count were higher in rats treated with aspirin compared to control. No significant alterations were observed in blood glucose level. After treatment with PMK and AMK, platelet count was decreased compared to control.
The significant reductions observed in the activity of ALT and AST levels in PMK and AMK treated animals compared to control.
Discussion
inflammatory process, in which vascular permeability increases and leukocyte migration occurs, involves inflammation mediators including neutrophil derived reactive oxygen species and free radicals, such as hydrogen peroxide, superoxide and the hydroxyl radical (Da Motta et al.1994), nitric oxide, prostaglandins (PG) and cytokines (Gualillo et al.2001). Polymorphonuclear leucocytes (Jain et al. 2001), which are the first cells to arrive at inflammatory site in the body, release free oxygen radicals (O2.) and free hydroxyl (OH.) radicals (Mc Cord and Roy 1982). Based on this, second phase may be explained by an inhibition of cyclooxygenase or exerting of antioxidant properties (Boughton-Smith et al. 1999). As the extract exhibited significant (P<0.05) protection against increase in paw volume at first hour as well as third hour of carrageenan injection, it might be having Histamine, 5-Hydroxy trptamine and PG synthesis inhibitory activity.
In order to explore the effect of antioxidant defenses on the acute inflammation process, in paw tissues, the levels of SOD, CAT and GSH and extent of LPO was evaluated. Preventive antioxidants, such as SOD and CAT enzymes are the first line endogenous defense mechanism against reactive oxygen species. GSH is a major low molecular weight scavenger of free radicals in the cytoplasm and an important inhibitor of free radical mediated LPO (Halliwell 1995). SOD scavenges the superoxide radicals O2, one of the ROS responsible for lipid peroxidation (Fridovich 1986). This reaction leads to increase in generation of peroxide radical H2O2 ., which is also capable of producing more oxidative damage (Das et al. 1997). CAT and other peroxidases further reduce H2O2. Hence, increase in SOD and CAT levels result in decreased LPO levels. Lipid peroxidation is a free radical mediated process, which has been implicated in a variety of diseased states. It involves the formation and propagation of lipid radicals, the uptake of oxygen and rearrangement of disulphide bonds in unsaturated lipids which eventually results
in destruction of membranes. Biological membranes are often rich in unsaturated fatty acids and bathed in oxygen and metal containing fluid. Therefore, membrane lipids are susceptible to peroxidative attack (Cheeman 1993).
According to this study, administration of PMK, AMK and Aspirin showed significant (P<0.05) increase in the SOD, CAT and GSH levels compared to the control animals. This suggests efficacy of PMK, AMK and aspirin in preventing free radical induced damage. Similarly, a significant (P<0.05) inhibition of lipid peroxidation by PMK, AMK was observed which may be attributed to prevention of, a decomposed product of disrupted membrane, calculated in terms of TBARS. This further supports protective effect of M. koenigii. Therefore, antioxidant activity of PMK and AMK as exhibited in the in vitro test gets clarified.
The various biochemical and haematological parameters investigated in this study are useful indices of evaluating the toxicity of plant extract in animals (Yakubu et al. 2008). The lower RBC count, Hb content and PCV after 11 days administration of aspirin suggest that it had induced anemia associated with leucocytosis as revealed by increased WBC, lymphocyte, neutrophils and basophils count (Raybak, 1992). PMK or AMK treated animals showed normal RBC count, Hb and PCV levels near to control group suggesting that rats were not anemic. Increase in WBC, lymphocyte, neutrophils and basophils count is a normal reaction of rats to foreign substances indicating stimulation of the immune system. Aspirin, PMK and AMK exerts a significant reduction in the platelets which indicates antiplatelet property and hence the usefulness in cardiovascular diseases (Aliyu et al. 2006). The significant reductions observed in the activity of ALT and AST indicate that PMK and AMK may not be harmful to the liver.
(N-H), 2974.33 μ (C-methyls), 1610.61 μ (aromatic system) suggesting presence of pyranocarbazole nucleus. The UV spectrum of AMK showing absorption peaks at 295.0, 256.50 and 246.50 nm also supports the presence of pyranocarbazole nucleus. (Bhattacharya et al. 1982; Reisch et al.1992; Joshi et al. 1970)
The presence of carbazole alkaloids, triterpenoids and flavonoids in Murrayakoenigii may be responsible for anti-inflammatory activity as all the three constituents have been reported to posses anti-inflammatory activity (Fernanda et al. 2004; Onasanwo and Elegbe 2000).
Fig.1. Free radical scavenging activity of PMK by DPPH method.
Fig.2 Total antioxidant activity of PMK by ammonium thiocyanate method.
Fig.3 Hydrogen peroxide scavenging activity of PMK.
Table 1 Effect of Murrayakoenigii on Carrageenan induced rat paw edema
0 20 40 60 80 100
25 50 100 200 400 800
%
Scav
eng
ed
Concentration in ppm
0 10 20 30 40 50 60 70 80
10 25 50 100 250 500
%
I
nhibi
ti
on
Concentration in ppm
0 20 40 60 80 100
10 25 50 100 250 500
%
I
nhi
bi
ti
on
Concentration in ppm
Treatment (mg/kg, p.o.)
Mean increase in paw volume (ml) % Decrease in paw volume
at 3rd h
1 h 2 h 3 h 4 h
Control 0.31±0.02 0.76±0.02 0.96±0.04 0.72±0.06 -
Aspirin 100 0.09±0.01* 0.16±0.02* 0.26±0.02* 0.16±0.01 72.91
PMK 100 0.12±0.04* 0.26±0.06* 0.40±0.08* 0.28±0.05* 58.30
PMK 300 0.21±0.06* 0.29±0.04* 0.32±0.02* 0.36±0.10* 66.60
n=5. The observations are mean ± SEM. *p < 0.05, compared to control (one-way ANOVA followed by Dunnett’s test).
Table 2: Effect of Murrayakoenigii on SOD, Catalase, GSH & LPO levels in Carrageenan- induced paw edema in rats.
n=5. The observations are mean ± SEM. *p< 0.05, compared to control (one-way ANOVA followed by Dunnett’s test).
Table 3: Effect of Murraya koenigii on the hematological parameters in carrageenan-induced inflammation in rat
Treatment (mg/kg )
Parameters
Control Aspirin 100
PMK 100
PMK 300
AMK 100
AMK 300
Glucose (mg dl-1) 116.56 ±2.23
102.53 ±3.26*
112.32 ±2.69*
115.39 ±3.5*
118.23 ±2.5*
109.45 ±1.6* Hemoglobin (g dl-1) 13.93±0.
75
6.2±1.59* 11.8±0.68* 12.1± 0.29*
12.23± 0.95*
14.29±1. 97* Red blood cells
(Million.m.mm)
4.96±0.0 2
2.96±0.04* 4.03±0.02* 4.26± 0.1*
4.03±0. 09*
4.12±0.0 8* Total leukocyte count
(cells m-1 mm-1)
3800±5. 4
5300±6.9* 4200±4.32* 4100± 4.8*
3900±5 .08*
4500±4. 4* Lymphocyte 26±2.09 46±1.65* 32±1.78* 39±2.65* 38±0.9 42±1.09
AMK 300 0.11±0.04* 0.22±0.02* 0.27±0.01* 0.25±0.02* 71.87
Treatment (mg/kg p.o.)
SOD
(U/g wet tissue)
Catalase
(U/g wet tissue)
GSH
(nmol/mg wet tissue)
LPO
(μM/mg wet tissue)
Control 52.07±4.22 3.558±0.20 2.117±0.09 2.27±0.03
Aspirin 100 105.5±2.32* 12.21±0.60* 7.696±0.52* 0.58±0.05*
PMK 100 70.07±1.39* 5.965±0.52* 3.841±0.16* 1.37±0.02*
PMK 300 95.74±7.17* 9.507±0.29* 5.49±0.08* 0.91±0.05*
AMK 100 85.86±4.13* 8.72±0.66* 4.541±0.41* 0.87±0.02*
8* * Neutrophils 39±1.56 55±2.12* 52±1.89* 63±0.68* 65±1.6
3*
77±3.36 * Basophils 2.5±0.98 1.12±0.65* 2.69±0.87* 3.12±
0.85*
2.79±0. 76*
3.42±1.2 4* Plateletcount
(lakhs.m.mm)
2.97±0.0 9
1.90±0.15* 2.22±0.55* 2.12± 0.87*
2.08±0. 98*
1.95±1.6
PCV (%) 44±0.69 28±0.60* 32±0.93* 40±0.5* 39±1.3 5*
42±4.86 *
AST (U/L) 122.32±
5.72
75.58±7.2* 62.5±3.6 5*
66.42± 5.78*
55.64±4. 90*
ALT (U/L) 129.13±
5.34
72.44 ±5.71*
65.29 ±5.32*
62.98 ±5.69*
52.33 ±4.65*
n=5. The observations are mean ± SEM. *p< 0.05, compared to control (one-way ANOVA followed by Dunnett’s test).
IV.
CONCLUSIONA significant % inhibition of paw edema and alterations in associated biochemical parameters by pet ether extract (PMK) of Murraya koenigii leaves and alkaloids (AMK) isolated from PMK at doses of 100 and 300 mg/kg/day, p.o., for 11days, suggest its usefulness as an anti-inflammatory agent.
V.
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